Orientadora: Eliana Gertrudes Macedo Lemos / Banca: Lúcia Maria Carareto Alves / Banca: Vanderlei Rodrigues / Resumo: Metabólitos secundários são compostos bioativos, com grande importância para a indústria farmacêutica e agropecuária, produzidos por certos grupos de microrganismos e plantas. Os policetídeos, que são sintetizados por complexos enzimáticos denominados policetídeos sintases (PKSs), desatacam-se entre os metabólitos secundários conhecidos e compõe a estrutura química básica de vários antibióticos. Todos os genes envolvidos na biossíntese de um policetídeo se encontram agrupados fisicamente no cromossomo, e contém genes que são altamente conservados, comumente chamados d~ pks mínima. Os métodos tradicionais para pesquisa de novas drogas, que envolvem o cultivo de microrganismos isolados do solo, não são mais tão promissores, devido à alta taxa de redescoberta de antibióticos já conhecidos, que chega a 99,9%, e à pequena parcela de microrganismos do solo que são cultiváveis pelas técnicas padrões de cultivo, cerca de 1 %. A Metagenômica é uma abordagem promissora que permite acessar o genoma desses organismos incultiváveis, pois consiste na extração de DNA diretamente do ambiente e construção de uma biblioteca com este genoma misto. Neste trabalho descrevemos a construção de uma biblioteca feita com DNA de alto peso molecular isolado diretamente de solo coletado sob arboreto de eucaliptos no Estado de São Paulo, Brasil. A biblioteca possui 9.320 clones e foi construída em vetor cosmídeo, com insertos de tamanho variando entre 30 e 45kb...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Secondary metabolites are bioactive compounds with great importance in the pharmaceutical and agriculture industries, procuced by a few groups of microrganisms and plants. The polyketides that are synthetized by enzimatic complexes, denominated polyketides synthases, outstand among the secondary known metabolites, which are part of the main structure of many antibiotics. Ali genes involved in the biosynthesis of antibiotics are found as clusters in the chromossome. The traditional methods for the research of new drugs that are made from microrganisms cultures isolated from the soil are not so promissing, due to the high rate of rediscorevy of already known species, reaching 99.9%. The other small piece of microrganisms are culturable by standards culture methods, reaching 1 % maximum. Metagenomics is a promissing approach that allows the access to genom of these organisms that are not culturable, as it is carried out by DNA extraction directly from the environment and construction of a mixed genomic library. In this work, we describe the construction of a library made from high molecular weight DNA isolated directly form the soi! undemeath a pinus forest in the State of São Paulo, Brazil. The library shows 9.320 dones and it was constructed in a cosmideo vector, with insert size ranging from 30 to 45 kb. Digestion with difterent restriction enzymes of cosmidial DNA randomly chosen allowed to visualize evident difterences in the restriction fragments among the clones, as does the possibility to determine the average insert size. The initial evaluation of the presence of genes involved in the biosynthesis of antibiotics synthesized by the enzymatic system PKS of kind I, was accomplished by the PCR amplification of clones from the library using specific primers. We studied 4.320 clones and the results suggest a great variety of these genes. The PCR products obtained were sequenced for the determination of identity of the amplified gene. / Mestre

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-28Bitstream added on 2014-06-13T20:47:49Z : No. of bitstreams: 1 schuch_v_me_jabo.pdf: 3089029 bytes, checksum: 0835ef08e49e97cfdf7ad571bdfc3671 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Metabólitos secundários são compostos bioativos, com grande importância para a indústria farmacêutica e agropecuária, produzidos por certos grupos de microrganismos e plantas. Os policetídeos, que são sintetizados por complexos enzimáticos denominados policetídeos sintases (PKSs), desatacam-se entre os metabólitos secundários conhecidos e compõe a estrutura química básica de vários antibióticos. Todos os genes envolvidos na biossíntese de um policetídeo se encontram agrupados fisicamente no cromossomo, e contém genes que são altamente conservados, comumente chamados d~ pks mínima. Os métodos tradicionais para pesquisa de novas drogas, que envolvem o cultivo de microrganismos isolados do solo, não são mais tão promissores, devido à alta taxa de redescoberta de antibióticos já conhecidos, que chega a 99,9%, e à pequena parcela de microrganismos do solo que são cultiváveis pelas técnicas padrões de cultivo, cerca de 1 %. A Metagenômica é uma abordagem promissora que permite acessar o genoma desses organismos incultiváveis, pois consiste na extração de DNA diretamente do ambiente e construção de uma biblioteca com este genoma misto. Neste trabalho descrevemos a construção de uma biblioteca feita com DNA de alto peso molecular isolado diretamente de solo coletado sob arboreto de eucaliptos no Estado de São Paulo, Brasil. A biblioteca possui 9.320 clones e foi construída em vetor cosmídeo, com insertos de tamanho variando entre 30 e 45kb... / Secondary metabolites are bioactive compounds with great importance in the pharmaceutical and agriculture industries, procuced by a few groups of microrganisms and plants. The polyketides that are synthetized by enzimatic complexes, denominated polyketides synthases, outstand among the secondary known metabolites, which are part of the main structure of many antibiotics. Ali genes involved in the biosynthesis of antibiotics are found as clusters in the chromossome. The traditional methods for the research of new drugs that are made from microrganisms cultures isolated from the soil are not so promissing, due to the high rate of rediscorevy of already known species, reaching 99.9%. The other small piece of microrganisms are culturable by standards culture methods, reaching 1 % maximum. Metagenomics is a promissing approach that allows the access to genom of these organisms that are not culturable, as it is carried out by DNA extraction directly from the environment and construction of a mixed genomic library. In this work, we describe the construction of a library made from high molecular weight DNA isolated directly form the soi! undemeath a pinus forest in the State of São Paulo, Brazil. The library shows 9.320 dones and it was constructed in a cosmideo vector, with insert size ranging from 30 to 45 kb. Digestion with difterent restriction enzymes of cosmidial DNA randomly chosen allowed to visualize evident difterences in the restriction fragments among the clones, as does the possibility to determine the average insert size. The initial evaluation of the presence of genes involved in the biosynthesis of antibiotics synthesized by the enzymatic system PKS of kind I, was accomplished by the PCR amplification of clones from the library using specific primers. We studied 4.320 clones and the results suggest a great variety of these genes. The PCR products obtained were sequenced for the determination of identity of the amplified gene.

Os microrganismos constituem dois terços da diversidade biológica na Terra, no entanto, muitos deles não podem ser cultivados por técnicas tradicionais. Portanto, o acesso a esta diversidade tem sido feita através da utilização de técnicas independentes de cultivo. Diante deste panorama, a metagenômica apresenta-se como uma alternativa, pois dispensa a necessidade de cultivo. Tal técnica possibilita inclusive a identificação e utilização do potencial metabólico destes organismos para o desenvolvimento de novos processos e produtos. Os polihidroxialcanoatos (PHAs) são poliésteres bacterianos, acumulados intracelularmente em forma de grânulos, cujas propriedades são similares a de alguns plásticos de origem petroquímica. O objetivo deste trabalho foi identificar e avaliar a diversidade de genes relacionados à produção de PHAs em bibliotecas metagenômicas de solo. A prospecção realizada resultou na identificação de clones contendo o gene phaC. De uma forma geral, pôde-se concluir que ainda há uma grande diversidade deste gene a ser descoberta no ambiente estudado. / Microorganisms constitute two third of the Earth\'s biological diversity, however, many of them cannot be cultured by standard techniques. Therefore, access to this diversity has been achieved through the use of culture-independent techniques. Facing this scenario, the metagenomic presents itself as an alternative, since it eliminates the need for cultivation. This technique also allows the identification and use of the metabolic pathways of these organisms to develop new processes and products. The polyhydroxyalkanoates (PHAs) are bacterial polyesters accumulated as granules, whose properties are similar to some plastics of petrochemical origin. The aim of this work was to identify and access the diversity of genes related to PHAs production in soil metagenomic libraries. The screening resulted in the identification of clones containing the phaC gene. In a general way, it was concluded that there is still a considerable diversity of this gene to be discovered in the study environment.

Os microrganismos constituem dois terços da diversidade biológica na Terra, no entanto, muitos deles não podem ser cultivados por técnicas tradicionais. Portanto, o acesso a esta diversidade tem sido feita através da utilização de técnicas independentes de cultivo. Diante deste panorama, a metagenômica apresenta-se como uma alternativa, pois dispensa a necessidade de cultivo. Tal técnica possibilita inclusive a identificação e utilização do potencial metabólico destes organismos para o desenvolvimento de novos processos e produtos. Os polihidroxialcanoatos (PHAs) são poliésteres bacterianos, acumulados intracelularmente em forma de grânulos, cujas propriedades são similares a de alguns plásticos de origem petroquímica. O objetivo deste trabalho foi identificar e avaliar a diversidade de genes relacionados à produção de PHAs em bibliotecas metagenômicas de solo. A prospecção realizada resultou na identificação de clones contendo o gene phaC. De uma forma geral, pôde-se concluir que ainda há uma grande diversidade deste gene a ser descoberta no ambiente estudado. / Microorganisms constitute two third of the Earth\'s biological diversity, however, many of them cannot be cultured by standard techniques. Therefore, access to this diversity has been achieved through the use of culture-independent techniques. Facing this scenario, the metagenomic presents itself as an alternative, since it eliminates the need for cultivation. This technique also allows the identification and use of the metabolic pathways of these organisms to develop new processes and products. The polyhydroxyalkanoates (PHAs) are bacterial polyesters accumulated as granules, whose properties are similar to some plastics of petrochemical origin. The aim of this work was to identify and access the diversity of genes related to PHAs production in soil metagenomic libraries. The screening resulted in the identification of clones containing the phaC gene. In a general way, it was concluded that there is still a considerable diversity of this gene to be discovered in the study environment.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / The use of conventional culture-based approach results in vast majority of microbes (90 - 99%) unaccounted for. However, over the past years, the use of metagenomics, which is a culture-independent comprehensive approach has enabled researchers to access nearly 100% of the microbiome. In this study, three hot springs (44 – 70 oC) in Limpopo province of South Africa were investigated as potential sources of genes encoding for DNA-manipulating enzymes (DNA polymerase, DNA ligase and endonuclease), which are central in genetic engineering. They are usually grouped into four broad classes (nucleases, ligases, polymerases and modifying enzymes) depending on the type of the reaction they catalyze. Accordingly, hot spring metagenomic DNA was successfully extracted using modified SDS-CTAB method involving gel purification and electroelution. Consequently, a portion of the extracted metagenomic DNA was used for sequencing and another for fosmid library construction. Sequencing was done using Illumina MiSeq next generation sequencing platform and sequence data analyzed and de novo-assembled using CLC Genomic Workbench, which resulted in 5 681 662 reads and 7 338 contigs. A metagenome expression fosmid library of approximately 2.16 x 103 clones was also constructed using CopyControl™ HTP Fosmid Library Production Kit with pCC2FOS™ Vector. A BLAST algorithm in NCBI revealed 57 distinct genes for DNA polymerase, 29 genes for DNA ligase and more than 100 genes for endonuclease II enzymes. Hence, three genes related to thermophiles representing genes for DNA polymerase, DNA ligase and endonuclease II were selected. Accordingly, the three genes were codon-optimized, synthesized and successfully cloned into pET- 30a (+) and overexpressed in Escherichia coli BL21 (DE3) by inducing with 0.5 mM IPTG and incubating overnight at 16ºC. The cells were lysed using B-PER Reagent, protein extracted and purified using AKTA start protein purification system and purity of 85- 95 % was achieved. From this study, it can be concluded that metagenomics as an approach, can be used to mine for putative DNA-manipulating enzymes from hot spring metagenome. Besides, further study should be conducted to formulate the developed DNA-manipulating enzymes and study the practical application and chart way for commercialization. Moreover, the constructed fosmid library could also be screened for potentially novel thermo-stable biomolecules of industrial and therapeutic importance.

Metabolic engineering has enabled studying microorganisms by the modification of their genetic material and analysis of their metabolism for the isolation of microbial strains capable of producing high yields of high value chemicals and biofuels. In this research, novel tools were developed to improve genetic engineering of microbial cells. In this matter, λ-PCR (lambda-PCR) was developed enabling the construction of plasmid DNA. This technique allows DNA assembly and manipulation (insertion, substitution and/or deletion) at any location of a vector. λ-PCR addresses the need for an easy, highly-efficient, rapid and inexpensive tool for genetic engineering and overcoming limitations encountered with traditional techniques. Then, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system (in vitro) in order to modulate protein expression in biosynthetic pathways. The ability of the sRNAs to regulate mRNA expression with statistical significance was demonstrated. Up to 70% decrease in protein expression level was achieved by targeting specific secondary structures of the mRNA with antisense binding regions of the sRNA. Most importantly, a sRNA was identified capable of protein overexpression by up to 65%. An understanding of its mechanism showed that its mRNA target region(s) likely lead to occlusion of RNase E binding. This mechanism was translated for expression of a diaphorase enzyme, which has relevance to synthetic biology and metabolic engineering in in vitro systems. Results were successful, showing a greater than 75% increase in diaphorase expression in a cell-free protein synthesis reaction. Next, Raman spectroscopy was employed as a near real-time method for microbial phenotyping. Here, Raman spectroscopy was used in combination with chemometric analysis methods through RametrixTM Toolboxes to study the effects of environmental conditions (i.e. illumination, glucose, nitrate deprivation, acetate, sodium chloride and magnesium sulfate) on the phenotypic response of the cyanobacterium Synechocystis sp. PCC6803. The RametrixTM LITE Toolbox for MATLAB® enabled processing of Raman spectra and application of principal component analysis (PCA) and discriminant analysis of principal components (DAPC). Two studies were performed. PCA and DAPC produces distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on the (i) presence of glucose in the growth medium, (ii) illumination, (iii) nitrate limitation, and (iv) throughout a circadian rhythm growth cycle, in the first study. The second study focused on the phenotypic response based on (i) growth in presence of acetate, (ii) presence of high concentrations of sodium chloride and (iii) magnesium sulfate starvation. RametrixTM PRO was applied for the validation of the DAPC models through leave-one-out method that allowed calculation of prediction accuracy, sensitivity and selectivity for an unkown Raman spectrum. Statistical tests (ANOVA and pairwise comparison) were performed on Raman spectra to identify statistically relevant changes in Synechocystis phenotypes. Next, comparison between Raman data and standardized analytical methods (GF-FID, UPLC, spectrometric assays) was established. Overall, good correlation were obtained (R > 0.7). Finally, genomic DNA libraries were enriched to isolate a deoxynivalenol detoxifying enzyme. To do this, library fragments from microorganisms was generated through oligonucleotide primed polymerase chain reaction (DOP-PCR) and transformed in a DON-sensitive yeast strain. Rounds of subculture were performed in the presence of DON and ferulic acid in order to isolate a strain capable of enzymatic degradation of DON. / Doctor of Philosophy / Metabolic engineering is the use of genetic engineering to modify microorganisms in order to produce high yields of valuable commodity chemicals. The goal of this research is to develop new methods to improve genetic modification and selection of microbial cells. The specific objectives were to: (i) develop new tools for DNA assembly and manipulation, (ii) utilize small synthetic RNA to control protein expression level, (iii) use Raman spectroscopy to study phenotypic responses to environmental changes and (iv) enrich for microorganisms that detoxify dangerous toxins. First, a new technique for DNA assembly, named λ-PCR (lambda-PCR), was developed. This method allows the easy manipulation of plasmid DNA with high-efficiency and low-cost compared to traditional techniques. Second, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system in order to modulate (downregulate or overexpress) fluorescent protein expression. Next, Raman spectroscopy was used to assess phenotypic response of cyanobacterial cells to different environmental modifications (light settings, salts, sugar, etc…). Finally, genomic library was used to discover and characterize enzymes capable of degrading a mycotoxin.